SUMMARY Dengue virus is a mosquito-transmitted flavivirus that causes hundreds of millions of human infections world- wide each year. There are four serotypes of Dengue (DENV1-4) that co-circulate in hyperendemic regions. Primary infection by a single serotype results in febrile illness and subsequent lifelong immunity to that serotype. Secondary infections by heterotypic serotypes, however, can lead to severe shock syndrome and death. Severe Dengue disease is thought to be caused by antibodies that are elicited during primary infection that can bind multiple serotypes but not neutralize them, and facilitate entry and infection in Fc? receptor positive cells thus causing antibody-dependent enhancement (ADE) of infection. While a live-attenuated four-component chimeric vaccine was recently implemented in three countries, this vaccine did not protect nave individuals against symptomatic infection. There is therefore significant rationale to explore alternative platforms such as subunit vaccines (immunogens) as either next-generation primary vaccines or as boosting agents to improve existing live attenuated virus vaccines. Furthermore, identification and development of DENV immunogens that elicit broadly neutralizing antibody (bNAb) responses is highly desirable. DENV E glycoprotein domain III (EDIII) is a small (~100 residue) ?-sandwich domain that is an attractive candidate for immunogen design, since many bNAbs target this region. However, immunodominant regions of this domain lie outside of critical neutralization epitopes, which has hampered its advancement as a subunit vaccine. We have developed a phage display platform for EDIII immunogen engineering and used it to produce resurfaced EDIIIs (rsDIIIs) in which non-productive epitopes are masked by mutation. Our structure-based design strategy involves production and screening of EDIII-based phage libraries in which residues that are not involved in interactions with model bNAbs are varied with restricted amino acid diversity. This approach has yielded a panel of rsDIIIs that exhibit reactivity toward a prototypic bNAb, thus maintaining the conformational integrity of the broadly susceptible epitope, but not toward type-specific or non-neutralizing antibodies. We propose to perform a full in vitro characterization of this immunogen panel for biochemical properties such as folding stability and binding affinity. Monovalent as well as bivalent and polyvalent immunogen presentation formats also will be explored. The most promising rsDIII immunogen candidates will be tested in mice for immunogenicity and ability to confer protective immunity in a serum transfer challenge experiment. The overarching goal of this work is to use innovative protein engineering approaches to overcome the traditional barriers with EDIII-based immunogens. This work will provide proof-of-concept in mice for novel subunit vaccine candidates against DENV, and possibly other flaviviruses of global concern.